Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Many endospore-forming bacteria embed alanine racemases into their spore coats and these enzymes are thought to convert the L-alanine germinant into D-alanine, a spore germination inhibitor. C. difficile packages the Alr2 alanine racemase into the spore. Here, I describe my findings that alr2 mutant spores more readily germinate in response to L-alanine as a co-germinant. Surprisingly, D-alanine also functioned as a co-germinant. Finally, I demonstrated that L- and D-serine are also co-germinants for C. difficile spores and C. difficile Alr2 can accommodate both alanine and serine as substrates. During the analysis on Alr2, I found that D-alanine can function as a co-germinant for C. difficile spores at 37 oC but not at 25 oC. Because most germination assays are conducted at room temperature, I tested the ability of other amino acids to act as co-germinants with TA at 37 oC and found that many amino acids previously categorized as non-co-germinants are actually co-germinants at 37 oC. Even though C. difficile spore germination is known to require an amino acid co-germinant, the amino acid spore germinant receptor was unknown. In search of the amino acid germinant receptor, I used EMS mutagenesis as a strategy to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, I identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, I identified non-isogenic mutations in yabG. For C. difficile, the YabG protease is critical for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A defined yabG mutant exacerbated the EMS mutant phenotype. Moreover, I found that various mutations in cspA caused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, my study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants during C. difficile spore germination and that two pseudoproteases (CspC and CspA) function as the C. difficile germinant receptors.
